1. Field of the Invention
This invention relates to a method of detecting expression of isoenzymes of cytochrome P450 (CYP450) and Phase II conjugating enzymes in the rat. More specifically, this invention relates to specific 5' and 3' specific oligonucleotide primers, as well as a method of using the same with reverse transcriptase-polymerase chain reaction (RT-PCR) to detect mRNA expression of the major isoenzymes of CYP450 and fatty acyl-CoA oxidase in the rat. This invention includes the development of an in vitro culture system using rat hepatocytes which has been optimized for expression of both cytochrome P450 and Phase II conjugating enzymes.
2. Description of the Prior Art
The cytochrome P450 mixed function oxidases (MFO) are a group of enzymes which are predominantly expressed within the liver, kidney, lung and intestine of mammalian species where they play an important role in the oxidative metabolism of both endogenous and exogenous (xenobiotic) compounds. The role of the various members of this enzyme superfamily in the metabolism of drugs and chemicals, as well as their potential role in the generation of toxic metabolites and chemical-induced carcinogenesis, is well established.
In particular, induction of specific CYP450 isoforms has been associated with drug-drug interactions in humans and increases in liver weight, proliferation of the endoplasmic reticulum and non-genotoxic liver carcinogenicity and tumorigenicity in rodents. Guengerich, Cancer Res 48:2946-2954, 1988; Lake, Ann Rev Pharmacol Toxical 35: 483-507, 1995; Hankinson, Ann Rev Pharmacol Toxicol 35:307-340, 1995; Parkinson, Toxicol Pathol 24:45-57, 1996; and Kirby et al., Toxicol Pathol 24:458-467, 1996; and Burchell et al., Pharmacol Ther 43:261-289, 1989.
In addition to CYP450 enzymes, Phase II enzymes, which are involved in the conjugation of xenobiotics and their metabolites for excretion, have also been associated with the bioactivation of xenobiotics to toxic and tumorigenic metabolites. Parkinson, Casarett & Doull's Toxicology, The Basic Science of Poisons (Ed., Klaasen CD), 5th Edn, pp 113-186, McGraw-Hill, Inc., New York, 1996; Boberg, Cancer Res. 43:5163-5173, 1983; Curran et al., Endocrine Rev 12:135-150, 1991; Bock, Pharmacogenetics 4:209-218, 1994; and Monks, et al., Toxicol Appl Pharmacol 106:1-19, 1990.
Examples of these Phase II enzymes include uridine diphosphate-glucuronosyltransferases (UDPGT), glutathione-S-transferases (GST), and sulfotransferases (ST). Historically, in vivo models are commonly used for the study of chemical-induced enzyme expression and hepatotoxicity. However, this approach is costly, time consuming and requires large quantities of test material.
Several approaches have been used to monitor the regulation of cytochrome P450 (CYP450) enzymes following exposure to xenobiotics. The approach most often employed is to measure the enzymatic profiles of microsomal protein fractions using enzyme selective substrates. Although this technique is useful for the study of substrate specificities, enzyme kinetics, and metabolism of chemicals there are several disadvantages which can limit the application of this technique in assessing the biochemical regulation of these enzymes. Two important disadvantages are that CYP450 enzyme activity requires additional cofactors (e.g., requirement for the presence of heme) and can show non-selectivity for, or be inhibited by certain chemical substrates (e.g., ketoconazole and metyrapone), resulting in potentially misleading or inaccurate assessments of enzyme activity.
Today, the chemical industry (Pharmaceutical and Chemical manufacturers) recognize the value in developing in vitro techniques to assess the safety and efficacy of drugs and chemicals at an early stage of development. In vitro techniques most commonly used to study the expression of hepatic metabolizing enzymes and cytotoxicity include precision-cut liver slices (Brendel et al., Methods in Toxicology (Ed. Tyson and Frazier), Vol 1 A. pp 222-243, Academic Press Inc. NY., 1993; and Gandolfi et al., Toxicol. Pathol. 24:58-61, 1996), primary cultures of hepatocytes and immortalized cell lines. Donato et al., In Vitro Cell Develop Biol 30A:574-580, 1994; and MacDonald et al., Human and Exp Toxicol 13:439-444, 1994.
However, without exception, these in vitro systems have limitations in their applications. For example, continuously dividing cell lines fail to preserve their ability to express or induce specific Phase I and II metabolizing enzymes, resulting in enzymatic activities which are either absent or too low to be measured. In addition, a loss of more than 50% of the total metabolizing enzyme levels have been reported within 24 hr of culture using non-dividing whole cell systems (e.g., precision-cut tissue slices and primary cell culture systems). Guzelian et al., Drug Metabol Rev 10:793-809, 1989; Waxman et al., Biochem J. 271:113-119, 1990; Paine, Chem Biol Interac 747:1-31, 1990; and Dunn et al., Biotechnol Prog 7:237-245, 1991.
A number of reports have demonstrated that primary hepatocytes cultured under conditions which restore normal cell's morphology and liver specific gene expression, can respond to xenobiotics with induction of specifically inducible CYP450 enzymes to levels comparable with those achieved in vivo. Bissel et al., Ann NY Acad Sci 349:85-98, 1980; Isom et al., J Cell Biol 105:2877-2885, 1987; Ben-Ze'ev et al., Proc Natl Acad Sci 85:2161-2165, 1988; Schuetz et al., J Cell Physiol 134:309-323, 1988; Musat et al., Hepatology 18:198-205, 1993; Arterburn et al., Hepatology 21:175-187, 1995; Kocarek et al., Mol Pharmacol 43:328-334, 1992; Sidhu et al., In Vitro Toxicol 7:225-242, 1994; and Zurlo et al., In Vitro Cell Develop Biol 32:211-220, 1996.
Examples of these cell culture conditions include the use of an extracellular matrix (ECM), Schuetz et al., J Cell Physiol 134:309-323, 1988, chemically defined culture media conditions and hepatocytes co-cultured with non-parenchymal cells. Begue et al., Hepatol 4:839-842, 1984; Rogiers et al., Biochem Pharmacol 40:1701-1706, 1990; Donato et al., In vitro Cell Develop Biol 30A:825-832, 1994; Guzelian et al., Proc Natl Acad Sci 85:9783-9787, 1988; Kocarek et al., Mol Pharmacol 38:440-444, 1990; Kocarek, In Vitro Cell Develop Biol 29A:62-66, 1992; and Kocarek et al., Biochem Pharmacol 48:1815-1822, 1994.
More recently, polyclonal and monoclonal antibodies have been generated against various isoenzymes of CYP450 found in rat, thereby allowing for a more selective and defined analysis of CYP450 expression, Parkinson et al., Meth. Enzymol. 206: 233-245, 1991. This approach to monitoring changes in CYP450 isoenzymes has many advantages over those which monitor enzyme activity, particularly with regard to enzymes which are regulated post-translationally (e.g., CYP2E1). However, the success of this approach is dependent on the quality and availability of reagents (e.g., polyclonal versus monoclonal antibodies) and may lack the specificity for determining enzyme subtype expression (e.g., CYP3A1 and CYP3A2). Moreover, the generation of antibodies and the measurement of proteins using Western immunoblot analysis are both labor intensive and time consuming and cannot be readily implemented when new enzymes are identified.
A number of reports have described use of ECM systems in studying the expression of liver-specific gene regulation in primary rat hepatocytes, Kocarek et al., Drug Metabol Dispos 23:415-421, 1995, Waxman et al., Biochem J 271:113-119, 1990, Sidhu et al., In Vitro Toxicol 7:225-242, 1994, and Zurlo et al., In Vitro Cell Develop Biol 32:211-220, 1996.
These reports indicate that 1) hepatocyte genes expression decreases markedly after cell isolation, 2) hepatocyte isolation procedures result in altered expression of CYP450 mRNAs, 3) hepatocyte gene expression is restored after several days in culture, 4) the maintenance and/or inducibility of one or more P450 isoenzymes are lost during the first 48 hr in culture, and 5) hepatocytes cultured on extracellular matrix-coated plates, and under specific culture conditions (e.g., modified Chee's medium) maintain a wide range of CYP450 isoenzyme expression and the activities of certain CYP450 enzymes are enhanced following exposure to CYP450 inducing agents.
Accordingly, the present inventors sought to develop a feasible, reproducible and simple in vitro system for evaluating xenobiotics as CYP450 enzyme inducers, which could be used for routine toxicology applications. The inventors have optimized the ECM system to study and monitor a broad range of liver biotransformation enzymes at the mRNA and protein levels. In contrast with the classical methods in which hepatocytes are seeded on ECM-coated dishes (e.g., collagen or Matrigel.RTM.), and then maintained on Matrigel.RTM. (sandwich or overlay), the present invention optimizes rat hepatocyte culture conditions for enzyme expression by suspending the hepatocytes in Matrigel.RTM. before seeding them in culture. Rat hepatocytes cultured under these culture conditions show an enhanced level of expression of a variety of liver metabolizing enzymes within 24 hr after initial plating in response to chemical inducers. This permits induction and maintenance in culture of liver-specific biotransformation enzyme genes for greater than seven days at both the mRNA and protein levels. More importantly, rat hepatocytes responded very similar to adult rat liver when exposed repeatedly to the same or similar types of inducing agents.
The inventors have demonstrated this improved rat hepatocyte culture system by utilizing prototypical CYP450 enzyme inducers, including PB as an inducer of the CYP2B subfamily, HC as an inducer of the CYP3A subfamily, 3MC as an inducer of the CYPlA subfamily and CLO as an inducer of the CYP4A subfamily (and FACO). In all of the in vitro experiments conducted, substantially complete agreement in the detection of CYP450 gene expression was found between the RT-PCR and western immunoblotting techniques.
These results show that CYP450 enzyme expression is regulated primarily at the mRNA level. Nebert, Biochem Pharmacol 47:25-37, 1994, Gonzalez et al., Pharmacogenetics 3:51-57, 1993 and Gonzalez et al., FASEB J 10:1112-1117, 1996. This is particularly true of the inducible forms the CYP450 enzymes, including CYP1A1/2, CYP2B1, CYP3A1/2 and CYP4A1. These enzymes are shown to be constitutively expressed at low or undetectable levels in this in vitro culture system but are markedly increased upon exposure to CYP450 inducing agents at both the mRNA and protein levels. The induction of these CYP450 enzymes involves transcriptional activation of the CYP450 genes, which may also involve message stabilization, resulting in an increase in the levels of mRNA and newly synthesized protein.
The expression of CYP2E1 mRNA, which is constitutively expressed in vitro at both the mRNA and protein levels, is known to be increased generally by protein stabilization, although mRNA stabilization and/or increased efficiency of mRNA translation may also be involved. Hunt et al., Xenobiotica 21:1621-1631, 1991 and Raucy et al., Critical Rev Toxicol 23:1-20, 1993.
CYP2C11, an adult male rat-specific CYP450 isoform, has been shown to be constitutively expressed and noninducible in male rat liver. Waxman et al., Biochem 88:6868-6871, 1991, Morohashi et al., FASEB J 10:1569-1577, 1996 and Prough et al., FASEB J 10:1369-1377, 1996. This enzyme has been shown to be partially regulated by androgenic hormones in vivo and the disruption of circulating levels of growth hormone patterns by xenobiotics can decrease expression of this enzyme.
As expected, CYP2C11 was found to be constitutively expressed at low levels and noninducible by the CYP450 inducing agents in male rat hepatocytes cultured under the present conditions.
In agreement with other investigators, we have observed that maintenance of 3MC-mediated induction of the CYP1A1 and CYP1A2 enzymes, PB-mediated induction of the CYP2B1 and CYP3A1 enzymes, HC-mediated induction of CYP3A1, CYP3A2, and CYP2B enzymes, and CLO-mediated induction of the CYP4A1 and FACO enzymes in rat hepatocytes, is critically dependent on the presence of an ECM (e.g., Matrigel.RTM.). Waxman et al., Biochem J 271:113-119, 1990, Jauregui et al., Xenobiotica 21:1091-1106, 1991, and Kocarek et al., Mol Pharmacol 43:328-334, 1992.
We have also observed that CYP3A1 and CYP3A2 are induced by both steroid and PB-type inducers and the induction is dependent on the concentration of the inducer in the medium. For example, treatment of hepatocyte cultures with hydrocortisone, at concentrations as low as 10 .mu.M, results in the induction of the CYP3A1 and CYP3A2 and CYP2B1 enzymes at mRNA level, whereas lower doses (e.g, 0.1 .mu.M) did not affect enzyme expression. Like CYP3A1, CYP2B1 mRNA levels increased in a dose-related manner in the presence of HC.
Another significant finding was the effect of CLO on hepatocytes cultured on Matrigel.RTM.. We have found, in agreement with in vivo studies, that CLO increased the expression of the CYP4A1, CYP2E1 and FACO enzymes at the mRNA level. Tugwood et al., EMBO J 11:433-439, 1992, Gulick et al., Proc Natl Acad Sci 91:11012-11016, 1994 and Johnson et al., FASEB J 10:1241-1248, 1996.
This finding suggests that activation of peroxisomal enzymes can occur in this system, that CLO causes an increase in the expression of multiple CYP450 enzymes and that CLO can up-regulate the expression of the CYP2E1 enzyme at the mRNA level. The effect of CLO on CYP2E1 expression has been observed following in vivo exposures.
Phase II enzymes are of considerable importance in toxicology. The pharmacological and toxicological effect of many reactive endogenous and exogenous compounds depends on their rate of formation and elimination, which involves Phase II conjugating enzymes such as UDPGT, GST and ST. The absence or presence of these enzymes has been associate with carcinogenicity and adverse reactions of certain drugs and chemicals.
To further characterize our in vitro system, we have studied the effects of liver enzyme inducers on the expression of UDPGT, GST-Ya and ST at the mRNA level. We have found that these enzymes are constitutively expressed and induced by prototypical CYP450 inducers, similar to the inducible CYP450 isoforms.
In contrast with CYP450 enzymes, Phase II enzyme expression has been scarcely studied in cell culture. However, several reports have shown that Phase II conjugating enzymes can be maintained in culture using modified culture conditions (Grant et al., Biochem Pharmacol 35:2979-2982, 1986, Vandenberghe et al., In vitro Cell Develop Biol 24:281-288, 1988 and Vandenberghe et al., Biochem Pharmacol 37:2481-2485, 1988), ECM (Kane et al., In vitro Cell Develop Biol 27A:953-960, 1996, Judah et al., Toxic Appl Pharmacol 125:27-33, 1994) or co-culture systems. Rogiers et al., Biochem Pharmacol 40:1701-1706, 1990.
We have found that these enzymes can be maintained and induced in hepatocytes cultured with Matrigel.RTM.. We have found that phenobarbital, as well as hydrocortisone, markedly enhance the expression of these enzymes. The regulation of these Phase II conjugating enzymes by glucocorticoids has been previously demonstrated in cultured rat hepatocytes. The data presented here support the potential utility of this culture system for assessing the induction of Phase II conjugating enzymes in the rat by new chemical entities (NCE).